CN105358571B

CN105358571B - Method

Info

Publication number: CN105358571B
Application number: CN201480037548.1A
Authority: CN
Inventors: I·艾尔曼亚维; D·西格莫德
Original assignee: CSL Behring AG
Current assignee: CSL Behring AG
Priority date: 2013-07-01
Filing date: 2014-07-01
Publication date: 2019-03-05
Anticipated expiration: 2034-07-01
Also published as: EP3016976A1; CN105358571A; WO2015000886A1; HK1220983A1; AU2014286268A1; JP6474798B2; AU2014286268B2; DK3016976T3; JP2016523877A; US10144774B2; KR102382662B1; ES2833100T3; KR20160027136A; EP3016976B1; US20160368970A1

Abstract

The present invention relates to the methods of improvement, are used for IgG purification, improve quality of the yield of the IgG of every liter of initial substance without damaging product.

Description

Method

The present invention relates to the improved methods for IgG purification.Especially described improved method is provided than in the early time The higher yield of method.

Background of invention

It is well known that immunoglobulin plays a significant role in the immune system of mammal.They pass through B- lymph Cell generates, and is found to be present in blood plasma, lymph and other body secretions.Immunoglobulin accounts for the big of plasma proteins in human body About 20%.The basic unit of immunoglobulin is the different tetramer, includes two heavy chains connected by disulfide bond and two light chains. Each of these chains has to form the effect of the variable region of the N-terminal at them of antigen binding site and responsible immunoglobulin Answer the constant region of subfunction.

In the presence of five kinds of major type of immunoglobulins with different biochemistry and physiological property: IgG (heavy chain), IgA (α), IgM (μ), IgD (δ) and IgE (ε).Human IgG represents the immunoglobulin of maximum abundance in blood plasma, and IgA is represented Main antibody type in the saliva of outer portion secretion such as respiratory tract and enteron aisle, tear and mucus.IgM is to recycle at present in the mankind Physically maximum antibody in system exists usually as the pentamer of basic immunoglobulin unit, and in course of infection Early stage occurs.

Initially, it is successfully used to from human plasma preparation IgG and prevents and treats various communicable diseases.The product of early stage is logical The method (alcohol grading) for crossing relative poor produces and includes impurity and aggregation to only being capable of intramuscular administration their degree. Improvement in terms of purification process has generated the IgG system for being suitable for intravenously applying due to the improved purity and quality of IgG preparation Agent (referred to as IVIG), and the preparation (referred to as SCIG) for subcutaneous administration has also been developed.

Commonly used in from the commercial run of plasma purification IgG based on designed by Cohn original method (Cohn E., et al., (1946), J Am Chem Soc, 68,459-475, Oncley et al., (1949), J Am Chem Soc, 71,541-550), It is traced back to the 1940s and depends on the cold fractional precipitation of plasma proteins.Ionic strength, pH and temperature by After gradually adding ethyl alcohol under the conditions of control, the plasma fraction method obtain therapeutically useful plasma proteins (coagulation factor, Albumin, immunoglobulin, Antithrombin III) enriched or concentrated fraction.Divide according to the ethyl alcohol through changing is developed The Kistler and Nitschmann of grade method (claim using Cohn classification from fraction II+III, I+II+III or comparable precipitate A For NA precipitating) obtain IgG (Kistler P and Nitschmann HS, (1952), Vox Sang, 7,414-424).

Short chain fatty acids (C6-C12) and α-has been displayed in the 1960s and beta-globin forms insoluble complex compound, And gamma globulin is then not easy to precipitate (Chanutin et al., (1960) Arch.Biochem.Biophys.89；218).

Steinbuch and Audran ((1969) Arch.Biochem.Biophys.134,279-294) is described using pungent A kind of IgG purification process of the hydrochlorate (i.e. caprylate, C8 saturated fatty acid) as precipitating reagent.With acetate buffer dilute with After reaching 4.8 final pH, non-immunoglobulin is precipitated from human plasma.After adding caprylate under vigorous stirring, obtain The solution of IgG must be enriched with.Purity and yield depend on amount, pH, the molar concentration of buffer and the coefficient of dilution of caprylate.

A large amount of non-immunoglobulin precipitatings are most preferably obtained in faintly acid pH rather than when being lower than pH4.5.Blood plasma is used 0.06M acetate buffer (pH 4.8) dilutes (2:1), then with 2.5 weight % octanoic acid salt treatment, to start to precipitate.It will The Batch absorption of supernatant on DEAE- cellulose is used to remove other impurity from separated IgG fraction.Steinbuch Et al. later stage work show the octanoic acid most protein that is present in Cohn ethanol faction III of precipitating and lipoprotein (and Immunoglobulin) purposes (Steinbuch et al., (1973), Prep.Biochem.3,363-373).

Identical method is applied to using the diluted human plasma of 2.16 weight % caprylate (Habeeb et al., (1984) Prep.Biochem.14,1-17).Habeeb et al. is followed using the caprylic acid precipition being classified on DEAE cellulose.It is produced The IgG from blood plasma substantially free of aggregation, fibrinolysin and plasminogen.In addition, obtained The anti-complement activity of IgG is low and relatively stable during storage.Therefore caprylate settling step is known as it is highly useful, and It is introduced into from many modernisms of blood plasma production IgG.

Other than alcohol, PEG and sad staging, also several chromatography methods are used to purify in conjunction with basic staging IVIG。

The most frequently used chromatographic process is ion-exchange chromatography, and it makes use of on protein and Ion Exchange Medium Surface distribution and charge density.Positively charged surface is presented in anion exchange resin.Charge density is specific to resin, and And it is typically independent of pH (within the working range of resin).Typical anionite will be combined with net negative charge Protein (i.e. when the pH of solution is higher than the isoelectric point of protein).In reality, the surface of protein does not present single Charge；But positive charge and negative electrical charge and neutral region is chimeric.Surface texture is specific to given protein, and It will be influenced by solution condition such as ionic strength and pH.The uniqueness can be developed to establish single protein and will combine anion Exchanger resin or the specified conditions being released from.By establishing these conditions, tool can be efficiently separated with (> 95%) in high yield There is the protein on only slightly different surface or charge property.

The improvement of chromatography resin carrier configuration aspects is so that large-scale chromatography becomes more conventional purification process Alternative solution in practice.Rigid resin allows the volume that quickly processing in (< 5 hours) is big, and high ligand density produces To the ability reinforced necessary to large volume processing.Simplify these factor branch combined with high yield, product purity and processing Hold purposes of the chromatography in extensive manufacture.

Especially, by cation and/or anion-exchange chromatography (sometimes in a separate step or series combination) For IgG purification in blood plasma or its fraction (as described in WO 99/64462).In most methods, with ion mode Using anion-exchange chromatography, i.e. use condition makes pollutant protein i.e. IgA, IgM, albumin, fibrinogen, turns Ferritin can combine, and the IgG is recycled in non-adsorbed fraction.

Sad salt precipitation method therewith after the combination of the ion-exchange chromatography for IgG purification be described in many publications In.Steinbuch and Audran ((1969) Arch Biochem Biophys 134,279-284) is described fine with DEAE IgG is further purified after dimension element precipitating caprylate.Lebing et al. (US 5,886,157), which is described, to be used in series for removing Remove two kinds of anion-exchange columns of IgM, IgA, albumin and other impurity.Lebing et al. combines the effect of caprylate mediation The two substantially reduces non-IgG protein by precipitating, thus using virus distribution (partition) and individual The inactivation of virus property of the package of fatty acid in incubation step.It is opened from reconstruct of the paste/sediment in pH4.2 containing IgG Begin and then added when the pH is adjusted to 5.2 caprylate so-called " pH fluctuation " importance be emphasised for for It is important for IgG enrichment procedures, it is therefore desirable to effectively reduce non-IgG protein.Then pass through mentioned ion exchange Chromatographic step reduces some other impurity such as IgA and IgM and the caprylate.

U.S. Patent No. 5,164,487 are related to the purposes of IgG preparation of the octanoic acid for manufacturing intravenous tolerization, the system Agent is free of aggregation, vaso-active substance and proteolytic enzyme.Method includes making containing the starting material of IgG and 0.4% to 1.5% Octanoic acid contact, then uses ion exchange or hydrophobic base chromatogram purification.

Due to the Continuous improvement of purification process, there is the differentiation of IgG product for many years.As mentioned above, first kind IgG Product is appropriate only for intramuscular use, because they lead to excessive adverse events in intravenous application.It is suitable for intravenously making First generation IgG product (IVIG) is prepared by the pepsin cleavage of starting material (Cohn fraction II), the cracking Purpose is removing immunoglobulin aggregates, and the aggregation leads to such as complement activation of serious adverse events, and makes The product of early stage can not intravenously be applied.It does not include column chromatography steps in the method.Have to be freeze-dried the product, To which period is kept stablizing and be dissolved immediately using preceding at a reasonable time.

The eighties mid-term introduce with low anti-complement activity and higher stability based on uncracked and unmodified Immunoglobulin molecules second generation IVIG, but it is still in the form of the product of freeze-drying.Include if the IVIG passes through The method of dry color chromatography step purifies.Pepsin cleavage is avoided, eliminates aggregation and particle by the precipitation method, and It is realized and is further purified by column chromatography ion-exchange.

It include special viral inaction steps for third generation IVIG, in method.Although heavy in purification process Shallow lake step especially eliminates many viruses, however with some patients of blood products treatment still by HIV infection, therefore further need Special step is taken to make inactivation of virus from these products and except virus removal.

The method continues to improve to further realize the more preferably purity of protein and quality, so that can get Stable liquid product to be prepared is possibly realized, and improves the safety and tolerance of these products for patients.This Subcutaneous preparations are developed outside.

IgG product is used in many clinical applications now.In addition to for treat primary or acquired immunodeficiency and Other than the conventional use of communicable disease, have been displayed these products also autoimmune disease and certain neurogenic diseases such as In the treatment of CIDP effectively.There is also the significant growths of the research quantitative aspects for the other therapeutical uses for being dedicated to IgG product. Therefore the increase in demand for IgG product.IgG product is the maximum blood plasma product of demand in world market now；In 2008, Market reaches about 82 tonnes (including 37 tons of U.S., European 21 tons and 17 tons of Asia), and has with about annual 7% rate Tendency (anticipated demand in 2013 is 110 tonnes) (The Worldwide Plasma Fractions Market of growth 2008.The Marketing Research version in Bureau, Inc., 2010 years April).Since human plasma is that valuable have The resource of limit, so requiring further improvement for the method from plasma purification IgG, to realize than current possible higher receipts Rate, while the quality of product is not damaged.Current method has the average yield of the IgG of every liter of blood plasma 3.7 to 4.2g, this only generation Table is present in most 55% of the IgG in blood plasma.

Invention summary

The present invention relates to the method for the improvement of IgG purification in other solution from blood plasma or comprising IgG and other oroteins, The method improves quality of the IgG yield of every liter of starting soln (preferably blood plasma) without damaging product.

The first aspect of the present invention is in purification process from comprising IgG, other immunoglobulins and/or other oroteins The method that the solution of pollutant increases IgG yield, including

(a) acid solution comprising IgG, other immunoglobulins and/or other oroteins pollutant, the acidity are provided Solution has the total protein concentration of 3.5 to 5.2 pH and at least 10g/l；

(b) solution is adjusted to 5.2 to 6.2 pH, while keeping below the conductivity of 1.5mS/cm；

(c) by the solution incubation at least 15 minutes；With

(d) any sediment is removed.

Preferably, the solution comprising IgG includes the antibody from blood plasma, it is highly preferred that the solution comprising IgG passes through people's blood The alcohol grading of slurry or poor (cryo-poor plasma) blood plasma of cryoprecipitate of people obtains.It is of the invention it is another preferably In embodiment, the solution comprising IgG is the supernatant from caprylic acid precipition.

Typically, the solution in step (a) has passed through super diafiltration clarification.Preferably, the pH of the solution in step (a) is 3.9 to 5.0, more preferable 3.9 to 4.6, even more preferably 3.9 to 4.3.Preferably, the protein concentration in step (a) be 10 to 50g/l, more preferable 20 to 25g/l.

In step (b), pH is adjusted to 5.2 to 6.2 pH, is preferably adjusted to 5.6 to 6.0 pH, is more preferably adjusted To 5.8 to 6.0 pH.Preferably, the pH in step (b) is adjusted through at least one polyhydroxylated amine compounds of addition come complete At.Preferably, the pH, which is adjusted, is higher than 250mM, more preferably higher than 500mM, even more preferably more than 750mM using concentration, most The preferably from about Tris of 1M is completed.Preferably, the pH adjusting was completed during at least 15 minutes time.In step (b) Conductivity be lower than 1.5mS/cm, preferably shorter than 1.2mS/cm, more preferably less than 1.0mS/cm, even more preferably less than 0.8mS/cm, most preferably 0.2 to 0.5mS/cm.Most preferably, the pH in step (b) is adjusted to pH 5.6 to 6.0, and described Conductivity is 0.2 to 0.5mS/cm.

Incubative time in step (c) is at most 72 hours, at most 48 hours, at most 24 hours, preferably up to 12 hours, More preferably up to 6 hours, most preferably 15 minutes to 90 minutes.Preferably, the incubation in step (c) is carried out in environment temperature.

Sediment in step (d) is preferably removed by depth-type filtration.For removing other methods of the sediment It is possible, such as other filter methods or centrifugation.

After step (d), other purification step such as chromatographic process, such as ion-exchange chromatography can be carried out.It is excellent Selection of land, ion-exchange chromatography step allow pollutant be bound to resin but do not allow IgG to be bound to resin under conditions of into Row.Preferably, the ion-exchange chromatography is anion-exchange chromatography.Preferably, in addition it is not adjusted to the conductance of solution Rate carries out the ion-exchange chromatography.

The method will also comprise viral inaction steps.Preferably, the viral inaction steps are low pH processing.It is preferred that Ground, the low pH processing for viral inaction steps carry out before step (a).

Detailed description of the invention

The present invention relates to pollute in purification process from comprising IgG antibody, other immunoglobulins and/or other oroteins The starting soln of object, the intermediate of preferably current process increase the method for IgG yield.It the described method comprises the following steps:

(a) acid solution comprising IgG, other immunoglobulins and/or other oroteins pollutant, the acidity are provided Solution have 3.5 to 5.2, preferably 4.0 to 5.0, more preferable 4.6 to 4.8 pH and at least 10g/l, preferably from about 10 to 50g/l, More preferably from about 10 to 40g/l, even more preferably 15 to 30g/l, the most preferably total protein concentration of 20-25g/l.

Composition comprising IgG can be any substance, be preferably the biofluid comprising IgG.Preferably, include The composition of IgG is blood, blood plasma or serum, or derives from blood, blood plasma or serum.However, also can be used in the present invention Other initial substances comprising IgG.For example, it is also possible to using method of the invention from other biofluids, in cell culture Clear liquid is enriched with IgG.

Composition comprising IgG can be paste or the solution of sediment, be preferred from the fraction of cold alcohol grading method, Such as (FII+III), (F I+II+II) or FIII, as described by the method for Cohn/Oncley et al. or its modification, or precipitating Object A (PPT-A), sediment B (PPT-B) and sediment G (PPT-G), as retouched in Kistler the and Nitschman method It states or its modification.

However, it is preferred that the solution comprising IgG be using octanoic acid, polyethylene glycol and/or ammonium sulfate precipitation method from above The ethanol faction sets out the solution or any containing in IgG of the intermediate solution or intermediate sedimentation object that obtain during IgG purification Mesosome.When from serum fraction, caprylic acid precipition is the preferred method for preparing the intermediate of enrichment IgG.It can will be sad It is added to midbody solution, such as is added in stabilized ethanol pellet, until the ultimate density of about 5% (w/w), such as Steinbuch and Audran ((1969) Arch.Biochem.Biophys.134,279-294) is described.Also it can be used higher The octanoic acid of concentration.The octanoic acid should be slowly added under the condition such as pH, conductivity and incubative time of restriction in environment temperature. If can in addition add calcium phosphate, followed by during other incubation using the octanoic acid of higher concentration.Pollution albumen The sediment of matter, lipid and sad salt form, while main immunoglobulin, the especially described IgG retain in the solution.It is heavy Protein, lipid and the caprylate in shallow lake can be by being filtered to remove in environment temperature (such as 18 DEG C to 26 DEG C).Such as it can be Depth-type filtration (such as using diatomite, but other filter aids also can be used) is used in the presence of filter aid.It can be used general Ground flow filtration carrys out filtering solution.It removes sediment however, being also contemplated within and makes the clear other methods of solution.Then can make through Clear solution is subjected to diafiltration/ultrafiltration to adjust pH, conductivity and protein concentration.

Preferably, starting soln is under acid condition described above from blood plasma or the process of serum fraction IgG purification Period is clarified by ultrafiltration/diafiltration and has been enriched the intermediate solution of immunoglobulin.

If by sediment be used as initial substance, can by by sediment in buffer settling flux several hours from The sediment (process fraction (process fraction) or secondary fraction (side fraction)) extracts the IgG.It is preferred that Ground, using having 1 to 15mS/cm, the solution of more preferable 5 to 15mS/cm conductivity carries out solution.For molten liquefied molten The pH of liquid is 3.5 to 6, preferably 4.0 to 5.5, more preferable 4.5 to 5.2, most preferably from about 4.8.For example, solutionization can use 0.2M acetate is carried out in pH 4.8.However, those skilled in the art can determine other suitable buffer.Buffer with The ratio of sediment can be about 1:5 to 1:10, but other ratios also can be used.Under vigorous stirring using suitable mixing Device carries out solutionization at least 2 hours, preferably at least 4 hours.

In the next step of method of the invention, the pH of the solution of step (a) is further adjusted to about 5.2 to 6.2, It is preferred that 5.4 to 6.0, more preferable 5.6 to 6.0, even more preferably 5.7 to 5.9, most preferably from about 5.8.The conductivity of the solution is only Stand on pH and for lower than 1.5mS/cm, such as 0.2 to 1.5mS/cm, preferably shorter than 1.0mS/cm, such as 0.2 to 1.0mS/cm, Even more preferably less than 0.8mS/cm, such as 0.2 to 0.8mS/cm, or even lower than 0.5mS/cm, most preferably 0.2 to 0.5mS/cm。

Preferably, the reagent for adjusting pH and conductivity is following or comprising following: amine compounds, preferably polyhydroxylated Amine compounds, such as 2- amino -1- ethyl alcohol (C₂H₇NO), bis- (ethoxy)-amine (C₄H₁₁NO₂), three (2- ethoxy) amine (C₆H₁₅NO₃), preferably (bis- (2- ethoxy)-amino-three (methylol)-methane) (C₈H₁₉NO₅), N, N- bis- (2- ethoxys) is sweet Propylhomoserin (C₆H₁₃NO₄), bis- (three (methylol) methylaminos) propane (C of 1,3-₁₁H₂₆N₂O₆), most preferably 2- amino -2- methylol - Propane -1,3- glycol (C₄H₁₁NO₃) (Tris alkali).

The present inventor has been advantageously discovered that, is caused using the polyhydroxylated amine compounds with or without carboxyl Low IgG during secondary pH adjusts (stabilizing IgG) loses.Protein pollutant is precipitated, while conductivity being kept It is constant in desired value, such as 0.2 to 0.5mS/cm.

In step (c), using suitable mixer then the solution that this includes IgG is incubated at least under vigorous stirring 15 minutes.Those skilled in the art will adjust the time required for the step according to for mixed method.Incubative time It can be at most 72 hours, preferably up to 48 hours, more preferable 12 to 24 hours, most preferably 15 minutes to 12 hours.However, also Other incubative times can be used；Technical staff will recognize for longer incubative time, it may be necessary to using lower Temperature, such as 4-8 DEG C, and for shorter incubative time, can choose environment temperature.

In step (d), remove the protein through precipitating that is formed in the pH transition period of step (c), for example, by Environment temperature (such as 18-26 DEG C) depth-type filtration.The depth-type filtration needs to add filter aid, as known to technicians like that. General flow filtration can be used and carry out filtering solution.It is also possible, however, to use other methods of the protein through precipitating are removed, Such as other filter methods, centrifugation.Technical staff sufficiently knows other suitable methods for removing sediment.The step is led Cause significantly reducing for IgM and IgA.

Then it can handle clarified solution so that IgG is further purified.A kind of preferred option is to pass through the solution By ion-exchange chromatography.Preferably, remaining IgA, IgM and other pollutants is being allowed to combine solution, while IgG is kept It is loaded on anionite under conditions of circulation.Preferably, clarified solution can be directly loaded on column.Have Sharp ground, there is no the needs for adjusting pH or conductivity.

Anionite for the step can be strong anion exchanger or weak anion exchanger.Preferably, The anionite includes anion exchange ligand such as quaternary ammonium, quaternary ammonium ethyl, diethylamino ethyl, trimethylamino Ethyl or dimethyl aminoethyl.It is highly preferred that the anionite is selected from DEAE Sepharose FF, Q- Sepharose (HP and FF), ANX Sepharose FF (low and high substituted), Capto Q, Capto Q XP, Capto DEAE, Source 30Q and 15Q, most preferably Fractogel DEAE and MPHQ.

It is preferable to allowing remaining IgA, IgM and other pollutants to be bound to the anionite Under conditions of loaded.Typically, two buffer solution systems (equilibration buffer 1 and 2) are generallyd use and are using forward horizontal stand institute Anionite is stated, equilibration buffer 2 is thus used before load.Equilibration buffer is adapted for used anion exchange The ordinary buffer liquid system of agent.The conductivity of the equilibration buffer 1 is 10 to 20mS/cm, more preferable 11 to 15mS/cm.pH It is 7 to 8, more preferable 7.1 to 7.5.The example of suitable buffer is phosphate or acetate buffer or combinations thereof.It is preferred that Ground, buffer are phosphate mixt (unitary and binary).

The other steps that may include in the method are inactivation of virus/removing step, at nanofiltration, solvent/detergent Reason, low pH processing or pasteurization.Technical staff will fully understand suitable inactivation of virus and removing method.These steps can be with Including any appropriate stage in method.

Typically, pharmaceutically acceptable excipient such as stabilization agent can be added.

Wherein a particularly preferred method example of the method for the invention of successful implementation is such method, including Cold alcohol grading and using sediment A (according to the intermediate of the basicity grade of Kistler and Nitschmann point) or according to Cohn etc. The sediment II+III (PPT II+III) that people the obtains and/or sediment I+ obtained according to the modification of Kistler-Cohn method II+III。

Intermediate is prepared basically by steps of processing:

(1) sad (OA) classification,

(2) low pH is incubated,

(3) pH- conversion and depth-type filtration,

(4) anion-exchange chromatography,

(5) virus filtration, and

(6) it is concentrated by ultrafiltration/diafiltration.

Modification according to the method for the present invention includes one or more below

(i) pass through the prepurification of change pH (pH conversion) before depth-type filtration.The step low-down conductivity into Row, to precipitate the major part of IgA and IgM, and minimizes the co-precipitation of IgG before refining chromatographic step；

(ii) change buffer solution system for anion-exchange chromatography step.

Advantageously, (preferably) pH is carried out using the trihydroxy aminomethane buffer solution for the conductivity for not increasing solution to turn It changes；Therefore can will after depth-type filtration generated filtrate be directly loaded to for example, by using phosphate buffer suitably On the anion-exchange column of balance.Low conductivity for reducing the precipitating of IgG in this step for being important.

Using both modifications in the method, IgG yield increases at least compared with the same procedure without these modifications 5%.

Embodiment

The present invention will be illustrated now.Embodiment, which is intended to illustrate, to be not intended to limit the present invention.With reference to the following drawings:

Fig. 1: pH adjusts the effect to Protein yield (light grey column) and IgG yield (black column) in display step (b) Histogram.

Fig. 2: for the display initial substance different for three kinds, for converting pH to the concentration pair of the Tris of pH5.8 The histogram of IgG yield (black column) or the effect of loss (grey column): (a) the initial substance NA of the blood plasma from recycling PPT, (b) the initial substance NA PPT from source blood plasma, (c) from the PPT II+III of source blood plasma.

Embodiment 1

(a) according to the art methods ((1969) of Steinbuch and Audran Arch.Biochem.Biophys.134,279-294)。

The NA sediment (PPT) of a part freezing is resuspended in enough sodium acetate buffers, by NA PPT suspension Several hours are stirred until overwhelming majority IgG is dissolved in environment temperature, while pH is held constant at 4.8.

By the way that sad (OA) is added to suspension and then incubates progress degreasing in 240 minutes.Then calcium phosphate is added simultaneously By 60 to 90 minutes of suspension stirring in addition.By other dirts of the protein of precipitating, lipid or lipoprotein complexes and precipitating Dye object is removed by filtration under these conditions.

So that OA- filtrate is subjected to ultrafiltration to reach 3% protein concentration, is then percolated relative to water for injection (WFI).? PH is continuously converted to pH4.1 with 0.2mol/L HCl during diafiltration.Protein solution is diluted to 20g/L with WFI after diafiltration PH is adjusted to pH 4.0 ± 0.1 by protein, and adds 23.5mg/kg polysorbate 80 (P80).After filtering and filtering After washing, pH 4.0 is incubated at 37 DEG C with the protein concentration of about 20g/L and carries out several hours.Then pH 4 is incubated Substance is cooled to room temperature.

PH is increased into pH 6.5 with NaOH, is then incubated about 90 minutes.PH adjust and incubation be removed by filtration IgA and The major part of IgM.The impurity of precipitating is removed by filtration.After adjusting conductivity, by clear solution on-line filtration (filtered inline) is simultaneously applied on column, column 10mM sodium acetate, pH6.5, is adjusted and is balanced, filled with strong yin Ion-exchanger (Macroprep High Performance；MPHQ).Under prescribed conditions, remaining IgA and IgM and other Protein impurities are bound to the anion exchange (AIEX) resin, while finding IgG circulating and washing in fraction.It is collecting The pH of period, AIEX circulation and washing fraction comprising IgG are reduced to and are maintained at pH 4.8 ± 0.1.

By 0.1 μm of pre-filtering, then the removing of virus is realized in online nanofiltration.It is seeped using poly (ether sulfone) film and relative to WFI Viral filtrate is concentrated into 2-3% protein by filter.During diafiltration, pH is continuously converted to pH 4.1.Solution is concentrated later To the protein content of 105-135g/L.Bulk pharmaceutical chemicals are diluted to every liter of 100g IgG, it is (final with 250mmol/L L-PROLINE Concentration) stablize and pH is maintained at 4.80 ± 0.10.Preparaton ontology (formulated bulk) is to be filtered by 0.2 μm of film The particle of device filtering.

(b) include pH conversion and optimization chromatography buffer solution system improvement method

It is improved described in (a) by implementing pH switch process and optimizing chromatographic buffer solution system according to the present invention Method.

Processing NA PPT as described above, including OA precipitating and low pH inactivation of virus.

Then the pH of low pH 4 solution incubated is adjusted to pH5.8 using 1M Tris buffer.The pH is adjusted through 90 It carries out during the time of minute, is then incubated 90 minutes in environment temperature.Then it is removed by filtration and is formed by sediment.It will Such as about 5.8 pH and about 0.2- are held constant at described in pH and conductivity step (b) as claimed in claim 1 The conductivity of 0.5mS/cm.

(5mM phosphate+10mM is being adjusted and used using phosphate buffer (0.12M phosphate, pH 7.3 ± 0.2) Acetate, pH 6.0 ± 0.1) balance after, will be filled with strong anion exchanger (Macroprep High Performance；MPHQ chromatographic column) is resin-carried with linear flow rate use≤every liter of 180g protein of 70-130cm/h.? Under specified criteria, remaining IgA and IgM and other oroteins impurity are bound to anion exchange (AIEX) resin, while IgG is found in circulation and washing fraction.During collection, the pH of AIEX comprising IgG circulation and washing fraction be reduced to and It is maintained at pH 4.8 ± 0.1.

Following step is carried out as described in (a).

Yield comparison (method of existing method comparison improvement)

The comparison of two methods about the Protein yield from identical starting intermediates is shown in table 1 in 3.It will be low IgG yield after pH incubation (pH 4) is set as 100%.

Table 1: the comparison between the prior art and the method for improvement --- the initial substance NA generated by the blood plasma recycled

Step	Art methods	The method of improvement
			Plasma equivalent (L)	11195	239.6
After low pH is incubated
			Protein yield (%)	100	100
After pH- conversion and filtering
			Protein yield (%)	90.7	94.5
After chromatography
			Protein yield (%)	78	84
After this body preparation (bulk formulation)
			Protein yield (%)	77.6	83.3

Table 2: the comparison between the prior art and the method for improvement --- the initial substance NA generated by source blood plasma

Table 3: the comparison between the prior art and the method for improvement --- the initial substance PPTII+III generated by source blood plasma

Step	Art methods	The method of improvement
			Plasma equivalent (L)	13494	304.2
After low pH is incubated
			Protein yield (%)	100	100
After pH- conversion and filtering
			Protein yield (%)	96	100
After chromatography
			Protein yield (%)	80	85
After this body preparation
			Protein yield (%)	79	84

It has been shown that increased as a result, there is no obvious in terms of the purity of product with yield as changing by method Change.

Embodiment 2

This example demonstrates compared with existing 6.5 pH conversion, adjusted using the different pH of Tris buffer to IgG The influence of yield.

The PPT-NA described in embodiment 1 generates the solution that low pH 4 is incubated as described above.Extract eight parts (every part of 1kg), and it is adjusted to 1M Tris buffer to 5.2,5.4,5.6,5.8,6.0,6.2,6.4 pH.According to existing Technical method adjusts last a pH using 0.2M NaOH.PH adjusting was carried out during 90 minutes time, then as above It is literary described at environment temperature incubation 90 minutes.Then it is removed by filtration and is formed by sediment.Then filtrate is loaded to AIEX column, as described in embodiment 1 (b).Compare the protein and IgG yield of the solution through different loads.As a result shown in Figure 1. By in different pH conversion 1M Tris buffer be used for the 0.2M of art methods described in embodiment 1 (a) Influence of the NaOH relatively to IgG yield is shown, with the increased pH conversion (pH:5.2-6.2) of 1M Tris buffer is used, is made The minimization of loss of IgG yield.

Embodiment 3

This example shows the influences that the concentration of used Tris buffer is converted in the desired pH to 5.8.From The solution that low pH 4 is incubated sets out, and is respectively converted pH to 5.8 from 4 using 1M, 0.5M and 0.25M Tris buffer.

As a result shown in Figure 2.Independently of initial substance (source PPT-II+III, NA or the NA of recycling), IgG is lost with institute The increase of the molar concentration of the Tris buffer used and reduce.

Embodiment 4

This example demonstrates different reagents (including the amine compounds, preferably polyhydroxy for adjusting pH in the step (b) The amine compounds of change) influence to IgG yield.

Use following reagent: 2- amino -1- ethyl alcohol (C2H7NO), bis- (ethoxy)-amine (C4H11NO2), three (2- hydroxyl second Base) amine (C6H15NO3), preferably bis- (2- ethoxy)-amino-three (methylol)-methane (C8H19NO5), N, bis- (the 2- hydroxyl second of N- Base) glycine (C6H13NO4), bis- (three (methylol) methylaminos) propane (C11H26N2O6) of 1,3-, 2- amino -2- hydroxyl first The combination of base-propane -1,3- glycol (C4H11NO3) (Tris alkali) and these reagents.

The PPT-NA described in embodiment 1 generates the solution that low pH 4 is incubated as described above.Extract eight parts (every part of 1kg), and it is adjusted to using the 1M solution of mentioned reagent 5.75 to 5.85 pH.0.2M is used according to existing method NaOH adjusts last a pH.PH adjusting was carried out during 90 minutes time, then as described above in environment temperature It incubates 90 minutes.Then it is removed by filtration and is formed by sediment.Compare the IgG yield of different filtrates.

As a result it is shown in Table 4.It is as the result is shown described use for the pH conversion in step (b) different reagents (as 1M solution) IgG yield is influenced and using influence of the 0.2M NaOH to IgG yield as used in art methods Compare.The results show that increasing IgG yield using different amine compounds compared with art methods.It is particularly advantageous Use polyhydroxylated amine compounds.

Table 4: compared with art methods, between the prior art and the method using the improvement of different neutralization reagents Comparison --- the initial substance NA generated by source blood plasma

Claims

1. increasing IgG from the solution comprising IgG, other immunoglobulins and/or other oroteins pollutant in purification process The method of yield, including

(a) acid solution comprising IgG, other immunoglobulins and/or other oroteins pollutant, the acid solution are provided Total protein concentration with 3.5 to 5.2 pH and at least 10g/l；

(c) by the solution incubation at least 15 minutes；With

(d) any sediment is removed.

2. according to the method described in claim 1, wherein the solution comprising IgG includes the antibody from blood plasma.

3. according to claim 1 or method as claimed in claim 2, wherein the solution comprising IgG passes through the cold of human plasma or people The alcohol grading of the poor blood plasma of sediment obtains.

4. according to the method described in claim 3, wherein the solution comprising IgG is the supernatant from caprylic acid precipition.

5. according to the method described in claim 1, wherein the solution in step (a) has passed through super diafiltration clarification.

6. according to the method described in claim 1, wherein the pH of the solution in step (a) is 3.9 to 5.0.

7. according to the method described in claim 1, wherein the protein concentration in step (a) is 10 to 50g/l.

8. according to the method described in claim 1, the pH in step (b) is wherein adjusted to pH 5.6 to 6.0.

9. according to the method described in claim 1, wherein the pH adjusting in step (b) has by addition at least one or does not have There are the polyhydroxylated amine compounds of carboxyl to complete.

10. according to the method described in claim 1, wherein using the conductance in Tris regulating step (b) of the concentration higher than 250mM Rate.

11. according to the method described in claim 1, wherein the conductivity in step (b) is lower than 1.0mS/cm.

12. according to the method described in claim 1, the pH in step (b) is wherein adjusted to pH 5.6 to 6.0 and conductivity It is 0.2 to 0.5mS/cm.

13. according to the method described in claim 1, wherein the incubative time in step (c) is at most 72 hours.

14. according to the method described in claim 1, wherein the incubation in step (c) is carried out in environment temperature.

15. according to the method described in claim 1, wherein the sediment in step (d) is removed by depth-type filtration.

16. according to the method described in claim 1, allowing pollutant to be bound to resin wherein after step (d) but not permitting Perhaps IgG carries out ion-exchange chromatography step under conditions of being bound to resin.

17. according to the method for claim 16, wherein the ion-exchange chromatography is anion-exchange chromatography.

18. according to claim 16 or claim 17 described in method, wherein the ion-exchange chromatography is not to molten The conductivity of liquid carries out in the case where being additionally carried out adjusting.

19. according to the method described in claim 1, further including viral inaction steps.

20. according to the method for claim 19, wherein the viral inaction steps are low pH processing.

21. according to the method for claim 20, wherein the low pH processing carries out before step (a).